The present invention concerns improvement of fermented dairy products. In particular, the invention concerns a xcex2-galactosidase with transgalactosylating activity. More particularly, the invention concerns a xcex2-galactosidase isolated from Bifidobacterium bifidum where the C-terminal end of the protein has been deleted and the resulting truncated enzyme has higher transgalactosylating activity than hydrolase activity. When lactose is used as a substrate, galacto-oligosaccharides are products of the transgalactosylase activity. Galacto-oligosaccharides enhance growth of health-promoting Bifidobacterium that may be used in a number of applications in the dairy industry.
The genus Bifidobacterium is one of the most commonly used types of bacteria cultures in the dairy industry for fermenting a variety of dairy products. Ingestion of Bifidobacterium-containing products furthermore has a health-promoting effect. This effect is not only achieved by a lowered pH of the intestinal contents, but also by the ability of Bifidobacterium to repopulate the intestinal flora in individuals who have had their intestinal flora disturbed by, for example, intake of antibiotics. Bifidobacterium furthermore has the potential of outcompeting potential harmful intestinal micro-organisms.
Galacto-oligosaccharides are known to enhance the growth of Bifidobacterium. This effect is likely achieved through the unique ability of Bifidobacterium to exploit galacto-oligosaccharides as a carbon source. Dietary supplement of galacto-oligosaccharides is furthermore thought to have a number of long-term disease protecting effects. For example, galacto-oligosaccharide intake has been shown to be highly protective against development of colorectal cancer in rats (Wijnands, et al., 1999). There is therefore a great interest in developing cheap and efficient methods for producing galacto-oligosaccharides for use in the industry for improving dietary supplements and dairy products.
The enzyme xcex2-galactosidase (EC 3.2.1.23) usually hydrolyzes lactose to the monosaccharides D-glucose and D-galactose. In the normal enzyme reaction of xcex2-galactosidases, the enzyme hydrolyzes lactose and transiently binds the galactose monosaccharide in a galactose-enzyme complex that transfers galactose to the hydroxyl group of water, resulting in the liberation of D-galactose and D-glucose. However, at high lactos concentrations, some xcex2-galactosidases are able to transfer galactose to the hydroxyl groups of D-galactose or D-glucose in a process called transgalactosylation, whereby galacto-oligosaccharides are produced.
Enzymes capable of transgalactosylation have been isolated from a wide range of micro-organisms, including bacteria and yeasts. The observation that galacto-oligosaccharides enhance the growth of health-promoting Bifidobacterium has stimulated investigations of Bifidobacterium and their xcex2-galactosidase enzymes. Two DNA sequences of B. breve and B. longum xcex2-galactosidase genes have been deposited in GeneBank (accession numbers E5040 and AJ242596, respectively). Dumortier et al. (1994) have reported that B. bifidum DSM 20215 contains three xcex2-galactosidases and one of these enzymes has trans-galactosylating properties. However, no identification of the enzyme possessing this activity or any sequence of the enzyme or the corresponding gene from B. bifidum DSM 20215 has been published.
Production of galacto-oligosaccharides by the use of xcex2-galactosidases has been reported in several papers. For example, xcex2-galactosidase from E. coli has been shown to produce oligosaccharides at high lactose concentrations (0.5 M or approximately 20% lactose; Huber et al. 1976). Various thermophilic microorganisms have been shown to produce oligosaccharides at high temperatures and high lactose concentrations, e.g. Sterigmatomyces elviae can produce 39% oligosaccharides from 20% lactose at 60xc2x0 C. (onishi and Tanaka, 1995), and Saccharopolyspora rectivirgula can synthesize 41% oligosaccharides in 1.75 M lactose at 70xc2x0 C. (Nako et al., 1994).
However, the enzymes described above all have the drawbacks of requiring either high temperatures or high lactose concentrations or both in order to exhibit significant transgalactosylase activity. There is thus a need for developing cheaper and more efficient methods of producing galacto-oligosaccharides for use in the industry.
The present invention describes a new xcex2-galactosidase from Bifidobacterium bifidum. A truncated version of the enzyme has surprisingly been shown to have a high transgalactosylating activity. When the truncated enzyme, or a host cell expressing the recombinant truncated enzyme, is incubate with lactose under appropriate conditions, galacto-oligosaccharides are produced at a high efficiency. Presence of galacto-oligosaccharides in dairy products or other comestible products has the advantage of enhancing the growth of health-promoting Bifidobacterium in the product or in the intestinal flora of the consumer after intake of the product, or both.